Lettuce fortification through vitamin B12-producing bacteria - proof of concept study.

Background: Vitamin B₁₂ (cobalamin) can be produced de novo only by certain bacteria and archaea. It plays a crucial role in the health of animals and humans, which obtain it only through diet, mainly from animal products. This study aimed to identify endophytic bacterial strains capable of synthesizing vitamin B₁₂ and enriching edible plants with it as a potential solution for vitamin B₁₂ deficiency in vegetarians, vegans, and people with poor diets.

Results: An in silico genome analysis was performed on 66 bacterial genomes, including the reference strain Pseudomonas denitrificans ATCC 13867, a known vitamin B₁₂ producer. The genomes were analyzed using the Rapid Annotations using Subsystems Technology (RAST) server and the MetaCyc database to verify the presence and completeness of the vitamin B₁₂ metabolic pathway. The ability of the strains to produce vitamin B₁₂ was confirmed with a high-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis of pure culture extracts. Eleven strains produced detectable amounts of vitamin B₁₂ under tested conditions. The best performing candidates were further tested for their efficacy in producing vitamin B₁₂ in lettuce grown under sterile conditions on Murashige and Skoog (MS) medium with or without CoCl₂ supplementation. Methylobacterium sp. strain P1-11 produced detectable amounts of vitamin B₁₂ in planta: 1.654 and 2.559 μg per g of dry weight without and with CoCl₂ supplementation, respectively.

Conclusion: This is the first time a bacterial endophyte was used to produce vitamin B₁₂ in planta, suggesting that bacterial endophytes could be utilized to enhance the nutraceutical values of plant-based foods. © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.